monitoring sugar content of fruit juice using … monitoring sugar content of fruit juice using...
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Monitoring Sugar Content of Fruit Juice Using ACQUITY UPLC H-Class and BEH Amide Column Chemistry with Evaporative Light Scattering Detection (ELSD) Mark E. Benvenuti, Jennifer A. BurgessWaters Corporation, Milford, MA USA
IN T RO DU C T IO N
With today’s emphasis on healthy lifestyles, the consumption of pure fruit juices
rather than sugary soft drinks is considered to be beneficial for children and
adults alike. Fruit juice provides other health benefits, such as being a good source
of natural vitamins and antioxidants. For these reasons, fruit juices command
premium prices compared to other types of liquid refreshments and they can be
targets of adulteration.
There are many markers which can be used to identify potential adulteration
and these include amino acids, polyphenols and inositol content. Sugar content
itself is an important marker for a particular fruit juice. The European Fruit Juice
Association (www.aijn.org) provides information to its members regarding the
expected sugar content of different cultivar juices. Other researchers have also
published information regarding sugar content from different fruit juices, such as
Sanz et al.1 While there can be some variation in sugar content among different
cultivars, ratios of fructose, glucose and sucrose, the three most important food
sugars in fruit juice, tend to be constant as a function of fruit juice type.2 This
along with the presence of certain sugar alcohols such as sorbitol can also be used
to determine excursions in fruit juice quality.
In this application note, we will show data on sugar content for several fruit juices
along with their glucose/fructose ratios (G/F). Also we shall show the effect on these
ratios of spiking orange juice with high fructose corn syrup (HFCS) at various levels.
HFCS can be used as an adulterant for orange juice due to its low cost.3
WAT E R S SO LU T IO NS
ACQUITY UPLC H Class System
BEH Amide Column
ACQUITY UPLC Evaporative
Light Scattering (ELS) Detector
K E Y W O R D S
Fruit juice, polyphenol, sorbitol, fructose,
glucose, sucrose, sugar, cultivar, marker,
adulteration, HFCS
A P P L I C AT IO N B E N E F I T S
Using Waters® ACQUITY UPLC® H Class System
with BEH Amide Column Chemistry and
ELS detection provides many
benefits including:
■■ Run times of less than 10 minutes
for food sugars.
■■ ELS detection allows the option
of gradient elution leading to better
selectivity of saccharide analytes.
2Monitoring Sugar Content of Fruit Juice Using ACQUITY UPLC H-Class and BEH Amide Column Chemistry
E X P E R IM E N TA L
UPLC® conditions
UPLC system: ACQUITY UPLC H-Class
Runtime: 10.0 min
Column: ACQUITY UPLC
BEH Amide Column
1.7 µm, 2.1 x 100 mm
Column temp.: 85 °C
Mobile phase A: 0.05% Triethylamine
(TEA) dissolved in
water
Mobile phase B: 0.05% Triethylamine
(TEA) dissolved in
acetone
Injection volume: 3 µL
Time Flow rate %A %B (min) (mL/min)
1. Initial 0.25 10 90
2. 1.0 0.25 10 90
3. 10.0 0.25 50 50
4. 10.1 0.25 10 90
ELSD conditions
Nitrogen flow: 40 psi
Drift tube: 55 °C
Nebulizer: Cooling
Acquisition: 10 pts/sec
Gain: 50
Curve fit: Quadratic
Standard preparation
A stock solution was prepared by accurately weighing 0.5 to 0.55 g each of reagent
grade fructose, glucose, sucrose, maltose, and sorbitol and dissolving in 50 mL of
40:60 water/acetone. From this, a high concentration standard was prepared by
diluting 2 mL of stock to 10 mL with 40:60 water/acetone. Five further dilutions of
this standard were made to construct a six-point calibration curve using the standard
values listed in Table 1. Each standard was injected in triplicate.
Table 1. Standards 1 to 6, units are mg/L.
Standard Fructose Glucose Sucrose Maltose Sorbitol
1 2184.0 2068.0 2084.0 2196.0 2060.0
2 1092.0 1034.0 1042.0 1098.0 1030.0
3 436.8 413.6.0 416.8 439.2 412.0
4 218.4 206.8 208.4 219.6 206.0
5 109.2 103.4 104.2 109.8 103.0
6 87.4 82.7 83.4 87.8 82.4
Sample preparation
Samples of various fruit juices were purchased at a local market. Aliquots of
these juices were centrifuged at 4000 rpm for 30 minutes. The supernatant was
collected and diluted 1:50 with 40:60 water/acetone and injected in triplicate.
Samples of orange juice were spiked with varying levels of either 55 HFCS or 42 HFCS
and prepared as described above to determine the effect on the glucose /fructose and
fructose / sucrose (F/S) ratios (mean of three measurements).
3Monitoring Sugar Content of Fruit Juice Using ACQUITY UPLC H-Class and BEH Amide Column Chemistry
Minutes1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00
1- Fructose2- Sorbitol3- Glucose4- Sucrose5- Maltose
15
4
3
2
1300
LSU
Figure 1. Overlay of Standards 1 to 6.
R E SU LT S A N D D IS C U S S IO N
An overlay of the six standards, each containing the four sugars and sorbitol are shown in Figure 1. As shown in
Figure 1, baseline separation was achieved for the five compounds. ELS detection allows gradient elution which
enhances the separation of these compounds and allows the separation of higher polysaccharides if desired.4
The BEH Amide Column chemistry prevents the formation of Schiff bases and enamines which can cause loss of
reducing sugars. This robust column also allows the use of high pH modifiers and higher column temperatures
which promote anomer collapse leading to better peak shape and quantitation.4,5
4Monitoring Sugar Content of Fruit Juice Using ACQUITY UPLC H-Class and BEH Amide Column Chemistry
Using a quadratic fit for each of the calibration curves, coefficients of determination greater that 0.999 were
achieved for all compounds, as shown in Figure 2. Typical profiles for orange, white grape, pear, apple, and
pineapple juices that were purchased locally are shown in Figure 3. Differences in the sugar content for each
juice were observed and sorbitol was detected only in the pear and apple juice, as has been previously reported
in the literature.6 Table 2 lists the values for sugars found in the fruit juice matrices previously described.
Juice Fructose Glucose Sucrose SorbitolRatio
Glucose/ Fructose
Orange 1.60 1.51 3.86 ND 0.94
White Grape 6.65 6.19 ND ND 0.93
Pear 6.95 1.27 0.62 1.82 0.18
Apple 5.30 1.41 1.46 0.51 0.27
Pineapple 2.73 2.85 3.37 ND 1.04
Table 2. Sugar content of various fruit juices; units are g/100 mL; ND=not detected.
Minutes
1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00
1- Fructose2- Sorbitol3- Glucose4- Sucrose
300
LSU
200
LSU
200
LSU
300
LSU
300
LSU
1
4
3
1
3
2 3 4
1
2
3
4
13
4
1
Orange
Pineapple
Apple
Pear
White Grape
~ 1 LSU
Figure 3. Fruit juice profiles.
Figure 2. Calibration curves for sugars and sorbitol.
0
2x1064x106
0
2x1064x1066x106
0
2x106
4x106
0
2x1064x1066x106
0
2x106
4x106
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 1400.00 1600.00 1800.00 2000.00 2200.00
Fructose
Maltose
Sucrose
Glucose
Sorbitol
R2= 0.9996
R2= 0.9996
R2= 0.9995
R2= 0.9995
R2= 0.9994
Amount
5Monitoring Sugar Content of Fruit Juice Using ACQUITY UPLC H-Class and BEH Amide Column Chemistry
Minutes1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00
200
LSU
200
LSU
200
LSU
200
LSU
200
LSU
200
LSU
F
F
F
F
F
F
G
G
G
G
G
G
S
S
S
S
S
SOrange Juice- no spike
Orange Juice-0.25% HFCS spike
Orange Juice-0.5% HFCS spike
Orange Juice-1.0% HFCS spike
Orange Juice-2.5% HFCS spike
Orange Juice-5.0% HFCS spike
Figure 4. Samples of orange juice spiked with varying levels of 55% HFCS.
F/S Ratio 42% HFCS Spike F/S Ratio 55% HFCS Spike
G/F Ratio 55% HFCS SpikeG/F Ratio 42% HFCS Spike
Figure 5. G/F and F/S ratios for orange juice samples spiked with 42% and 55% HFCS respectively.
An additional experiment was undertaken to investigate the effect of spiking two separate samples of orange
juice each with varying levels of 42 and 55 (nominal % fructose) HFCS. The resulting chromatograms from
spiking 55 HFCS into orange juice from 0% to 5% are shown in Figure 4, and column graphs showing G/F and
F/S ratios for the two different types of HFCS are shown in Figure 5. The F/S ratios rose markedly as the orange
juice was diluted by either of the non-sucrose containing HFCS. Typical ratios of sucrose:glucose:fructose
are reported to be 2:1:1.3 This work suggests that monitoring the F/S ratio is a useful tool for detecting HFCS
additions to orange juice.
Waters Corporation34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
CO N C LU S IO NS
This application note has demonstrated a rapid method for
determining sugars in fruit juices with minimum sample
preparation. Use of Waters Technology for this purpose provides
benefits such as:
■■ Gradient elution at a higher temperature to improve selectivity
and collapse sugar anomers, for better peak shape.
■■ Quantification of sugars and determination of sugar ratios
as potential markers of adulteration.
■■ A method for rapid profiling of sugars and sugar alcohols
in different fruit juices.
■■ Confidence in sample quality for both incoming juice
as well as finished product.
Waters, UPLC, and ACQUITY UPLC, are registered trademarks of Waters Corporation. T he Science of What’s Possible is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.
©2012 Waters Corporation. Produced in the U.S.A.September 2012 720004404EN AG-PDF
References
1. Sanz et al. “Inositols and Carbohydrates in Different Fresh Fruit Juices” Food Chemistry. 87; (2004) pp. 326.
2. Ibid pp 325.
3. H S Lee, G A Coates. Quantitative Study of Free Sugars and Myo-Inositol in Citrus Juices by HPLC and a Literature Compilation. J Liquid Chromatography & Related Technologies. 23:14, 2124, 2000.
4. K Fountain et al. UPLC-MS of Carbohydrates. Waters Application Note, No. 720003212en, October, 2009.
5. C Hudalla et al. UPLC Analysis of Carbohydrates, Applications for Saccharide Analysis in Food and Beverage Products and Pharmaceutical Excipients. Waters Poster no. 72000321en, 2009.
6. Richmond et al. Analysis of Simple Sugars and Sorbitol in Fruit by High Performance Liquid Chromatography. , J Agric Food Chem. 29:1, 6, 1981.